Entry and infection of HIV is initiated by the binding of its envelope glycoprotein (Env) to a receptor (CD4) and a coreceptor (CCR5 or CXCR4), followed by fusion of its membrane with the membrane of the host cell. Understanding all aspects of these processes and discovering new or alternative ways to modulate it remain important areas research. Efforts have largely focused on the interactions of Env with CD4 and the coreceptors. In contrast, much less is known about the factors influencing the association of CD4 with the coreceptors preceding or during virus entry. Previously, we demonstrated a specific interaction between CD4 and CCR5 in cell lines and more importantly primary human cells as well. We have also observed and reported on the existence of oligomeric coreceptors. We propose to define the determinants of the CD4 and coreceptor interaction, the determinants of coreceptor oligomerization, and what their significance is in the Env-mediated fusion and virus entry steps of infection. Further, there is now a general consensus that clustering of a certain number of CD4 and coreceptor molecules is necessary for fusion. It has also been proposed that a possible mechanism for facilitating clustering is present and such a mechanism may be particularly important in primary cells where the numbers of CD4 and/or coreceptor molecules is limiting. Further, HIV Env can induce a variety of cellular signaling events and it is proposed that some of these cellular responses may contribute to the receptor clustering process and might also modulate early postentry stages of infection. We and others have observed that certain tyrosine kinase inhibitors can potently interfere with Env-mediated fusion and recently we have observed potent inhibition of both X4 and R5 virus infection of primary human macrophages by these reagents. Our data suggests that this inhibition is at an early stage of infection. Defining the significance of the signaling processes at play and how CD4 and coreceptors interact together and with Env during infection will enhance our understanding of the entry and pathogenesis of HIV, and perhaps facilitate discoveries of new ways for prevention and treatment. We propose to examine, certain specific signaling-related events occurring during entry and how CD4 and coreceptors interact and perhaps facilitate those events. Our Specific Aims are: 1) To define the molecular determinants of CD4 and coreceptor interaction; 2) To determine the significance of CXCR4 and CCR5 oligomerization for HIV-1 entry; and 3) To understand the mechanism of tyrosine kinase inhibitors on HIV-1 infection inhibition. We will utilize a variety of mutagenic approaches in conjunction with the use and characterization of a battery of non-human primate CD4 and CCR5 receptors; employ our assays of trimolecular complex formation and analysis; and our assays for measuring fusion and virus entry.